The proposed research involves investigations of the in vitro and in vivo metabolism of cyclophosphamide (CP) in an effort to define the role of metabolism in chemotherapy and toxicity of CP and understand the biochemical mechanisms by which CP produces specific toxicities, such as bladder toxicity, myelosuppression and the depression of hepatic drug metabolism. The overall objectives and the specific aims of these studies are to: (a) obtain a thorough understanding of both qualitative and quantitive aspects of enzymes involved in the metabolism (both activation and detoxification) of CP via various pathways; (b) elucidate the nature and extent of interactions between active metabolites of CP and tissue macromolecules, e.g. nucleic acids and proteins; (c) define the role of these interactions in chemotherapy and toxicity; (d) elucidate the biochemical mechanisms underlying specific toxic manifestations of CP; and (e) from the information gained from these studies, develop experimental and clinical approaches to reduce toxicity and thereby enhance chemotherapeutic index of CP. Compounds to be studied include analogs of CP, labeled and unlabeled CP and its metabolities (e.g. 4-hydroxy CP, 4-keto CP, carboxyphosphamide, phosphoramide mustard and other metabolites of CP), and specific drugs and anticancer agents used in conjuction with CP in the treatment and supportive care of cancer patients. Much of the proposed work will be performed (a) in vitro with 14(C-4)-CP, (3H-chloroethyl)-CP and some labeled metabolites of CP (e.g. 4-hydroxy CP, phosphoramide mustard), DNA (RNA or purine and pyrimidine-nucleoside polymers) and hepatic microsomes from mice and rats; (b) in vivo with labeled-CP in mice and rats, with and without tumors, to determine the in vivo macromolecular binding of CP and its relation to chemotherapy and toxicity; and (c) in vivo in rats and mice to elucidate and mechanism by which CP produces specific biochemical effects, e.g. glutathione depletion, lipid peroxidation. Particular methods to be used include: radiochemical techniques, thin layer chromatography, high pressure liquid chromatography, column chromatography, in vivo tumor chemotherapy, organic analytical techniques and other chemicals, biochemical and enzymological methods.